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. 2007 Dec;88(6):437–443. doi: 10.1111/j.1365-2613.2007.00554.x

Matrix metalloproteinase-2, -9 and -13 are involved in fibronectin degradation of rat lung granulomatous fibrosis caused by Angiostrongylus cantonensis

Cheng-Chin Hsu *, Shih-Chan Lai †
PMCID: PMC2517339  PMID: 18039280

Abstract

Pulmonary granuloma formation and fibrosis were experimentally induced in Sprague–Dawley strain rats by Angiostrongylus cantonensis. Increased protein levels of matrix metalloproteinase (MMP)-2, -9, -13 and the imbalance between these enzymes and metalloproteinase inhibitors, tissue inhibitors of MMPs (TIMP-1 and -2), occur during granulomatous fibrosis. Activation of proteolytic enzymes (MMP-2, -9 and -13) and fibronectin degradation occur simultaneously. Furthermore, the present study demonstrated that fibronectin avidly binds MMP-2, -9 or -13. Immunohistochemical observations also showed the localization of MMP-13, TIMP-1 and -2 within the infiltrating leucocytes. These results suggest that MMP-2, -9 and -13 may participate in the fibronectin degradation of A. cantonensis-induced granulomatous fibrosis.

Keywords: Angiostrongylus cantonensis, extracellular matrix, fibronectin, granuloma, inhibitors of matrix metalloproteinases, matrix metalloproteinases

The extracellular matrix (ECM) is an insoluble network of collagens, adhesive glycoproteins, and proteoglycans. It acts as a scaffold between tissues and modulates cellular functions, such as migration and proliferation (Shimizu & Shaw 1991). Fibronectin is a large glycoprotein of ECM, a disulphide-bonded dimer with a molecular weight of about 420 kDa. It is found in most body fluids and connective tissues, and is involved in cellular adhesion and phagocytosis (Ruoslahti et al. 1981). Adhesion is a mandatory step for leucocytes passing through the vascular endothelia and migrating to the site of inflammation. Pulmonary fibrosis is characterized by considerable disturbance of the tightly controlled equilibrium between synthesis and degradation of pulmonary ECM (Dunsmore & Rannels 1996).

Various molecules that influence inflammation, including cytokines (Alon et al. 1994) and degradative enzymes (Gilat et al. 1995), have been shown to interact with ECM moieties. The differential regulation of proteolytic enzymes that degrade ECM, such as matrix metalloproteinases (MMPs), can be grouped by substrate preference, shared structural motifs, or sequence homology. MMPs, also called matrixins, are the major family of enzymes involved in physiological and pathological degradation of ECM (Benyon & Arthur 2001). Some MMPs have a broad substrate specificity, degrading ECM proteoglycans, laminin, fibronectin, gelatin and the globular portion of basement membrane collagens (Matrisian 1990).

Previously, our laboratory has indicated that granulomatous fibrosis of rats infected with Angiostrongylus cantonensis is strongly associated with gelatinases, MMP-2 and -9 (Hsu et al. 2005). Also, tumour necrosis factor, interleukin-1β and MMPs were induced in pulmonary fibrosis (Tu & Lai 2006). In addition to MMP-2 and -9, the present study investigated whether MMP-13 was also associated with A. cantonensis-induced granulomatous fibrosis and, subsequently, whether fibronectin interacted with these MMPs (MMP-2, -9 and -13) in granulomatous fibrosis.

Materials and methods

Experimental animals

Five-week-old male rats of the Sprague–Dawley strain (specific-pathogen-free grade) were purchased from the National Laboratory Animal Center, Taipei, Taiwan. They were maintained with a 12-h-light:12-h-dark photoperiod, provided with commercial rodent food (Purina Laboratory Chow; Ralston-Purina, St Louis, MO, USA) and water ad libitum.

Larval preparation

The infective larvae (L3) of A. cantonensis originally obtained from a field of giant African snails (Achatina fulica) that were propagated for several months in our laboratory by cycling through rats and A. fulica. The larvae within tissues were recovered using a modification of the method of Parsons and Grieve (1990). Briefly, the shells were crushed, and the tissues were homogenized, digested in a pepsin-HCl solution (pH 1–2, 500 IU pepsin/g tissue), and incubated with agitation in a 37 °C waterbath for 2 h. Host cellular debris was removed from the digest by centrifugation at 1400 g for 10 min. The larvae in the sediment were collected by serial washing in double-distilled water and counted under a microscope.

Animal infection

In all, 90 male rats were randomly allocated to six groups (control, D15, D30, D45, D60 and D90) of 15 rats each. Food and water were witheld for 12 h before infection. The rats of experimental groups (D15, D30, D45, D60 and D90) were infected with 60 A. cantonensis larvae by oral inoculation and sacrificed on days 15, 30, 45, 60 and 90 postinoculation (PI). The control rats received water and were sacrificed on day 90 PI. The rats were sacrificed at different time points by anaesthetizing with ether, and the lungs were rapidly collected and frozen at −70 °C before use.

Antibodies

Goat anti-rat MMP-2 and -9 polyclonal antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Mouse anti-rat MMP-13 and tissue inhibitors of MMPs (TIMP)-1 polyclonal antibodies were purchased from NeoMarkers (Fremont, CA, USA). Rabbit anti-rat TIMP-2 polyclonal antibodies were purchased from NeoMarkers. Rabbit anti-rat fibronectin polyclonal antibody was kindly provided by Dr Cheng-Chin Hsu (Chung Shan Medical University, Taichung, Taiwan). Fibronectin was purified from rat plasma by affinity chromatography with gelatin-Sepharose 4B, and the antiserum was purified by treating it with immunoabsorbent material prepared from insolubilization of the fibronectin-free plasma fraction. The antiserum thus obtained was confirmed by immunoelectrophoretic analysis (Hsu et al. 1999). Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). HRP-conjugated rabbit anti-goat IgG and HRP-conjugated goat anti-rabbit IgG were purchased from R&D Systems.

Western blot analysis

Entire lung was homogenized in buffer containing 0.1% Triton X-100, 137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.5 mm KH2HPO4, a mixture of protease inhibitors (Sigma, St Louis, MO, USA) containing 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatinA, E-64, bestatin, leupeptin and aprotinin. The supernatant was recovered and stored at −20 °C until use. The protein contents (30 μg) of rat lung homogenate were diluted 1:1 in sample buffer (7.5% SDS, 2% glycerol, 10% bromophenol blue, β-mercaptoethanol and 0.5 m Tris–HCl, pH 6.8). Samples were centrifugated at 12,000 g for 10 min to remove debris. The protein was analysed using the method of Lai et al. (2004). Briefly, samples were submitted to SDS-polyacrylamide gel and electrotransferred to nitrocellulose membrane at a constant current of 190 mA for 90 min. The membrane was allowed to react with primary antibodies (anti-rat MMP-2, -9, -13, TIMP-1, -2 and fibronectin antibody) diluted 1:100 at 37 °C for 1 h. Then, the membrane was washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T), followed by incubation with HRP-conjugated secondary antibodies diluted 1:5000 at 37 °C for 1 h to detect the bound primary antibody. The reactive protein was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA). To confirm equivalent protein loading, membranes were stripped by incubation in 62.5 mm of Tris–HCl (pH 6.8), 2% SDS and 100 mm 2-mercaptoethanol at 55 °C, subsequently washed with PBS-T, and reprobed with anti-β-actin antibody (dilution 1:500).

Immunoprecipitation

To prevent non-specific adsorption, protein A/G agarose beads (Santa Cruz, CA, USA) were washed five times in 0.15 m PBS and then incubated with 5% bovine serum albumin (BSA) at 4 °C for 30 min. The solution was centrifugated twice at 10,000 g for 2 min to remove supernatant before use. Fibronectin antibodies were incubated with lung lysates (1 mg) at 4 °C overnight and collected by binding to protein A/G agarose beads. The beads were washed two times in dissociation buffer (0.5 m Tris–HCl, pH 8.0, 120 mm NaCl, 0.5% Triton X-100). Bound proteins were resolved by SDS-PAGE and the target protein (MMP-2, -9 or -13) association was determined by blotting.

Immunohistochemistry

The rat lungs were fixed separately in 10% neutral buffered formalin for 24 h, then dehydrated in a graded ethanol series (50%, 75%, 100%), cleared in xylene, and embedded in paraffin wax at 55 °C for 24 h. The histology was analysed using the method of Chen et al. (2004). For immuno-histochemistry, relatively thick (10 μm) serial sections of the wax-embedded lungs were cut, mounted on glass slides and dewaxed and rehydrated like the sections prepared for the histology. They were then treated with 3% H2O2 in methanol for 10 min, to inactivate any endogenous peroxidase, and washed three times, for 5 min each, with PBS. The sections were then blocked with 3% BSA at room temperature for 1 h, before being incubated with 1:50 dilution, in 1% BSA, of the primary antibodies (anti-rat MMP-13, TIMP-1 and -2 antibodies), at 37 °C for 1 h. After another three washes in PBS, the sections were incubated with the HRP-conjugated secondary antibodies diluted 1:100 in 1% BSA at 37 °C for 1 h, before three more washes in PBS. The sections were finally incubated, for 3 min at room temperature, with 3, 3’-diaminobenzidine (0.3 mg/ml) in 100 mm Tris (pH 7.5) containing 0.3 μl H2O2/ml. After a final three washes in PBS, the sections were mounted in 50% glycerol in PBS and examined under a light microscope.

Statistical analysis

The results obtained in this work were from triplicate experiments performed independently by identical methods. The results of tissue section experiment performed with six rats per group (total, 15 rats per group). Nine rats per group were the same in both Western blot analysis and immunoprecipitation. Results in the different groups of rats were compared using the non-parametric Kruskal–Wallis test followed by post-testing using Dunn’s multiple comparison of means. All results were presented as mean ± SD. P-values of <0.05 were considered statistically significant.

Results

The protein levels of MMP-2, -9, -13, TIMP-1, -2 and fibronectin

Using Western blot analysis, we performed immunoblots of rat lungs. The time-course studies of 94 kDa pro-MMP-9 bands were significantly increased during formation of granulomas. The 60 kDa immunopositive bands were detected by anti-MMP-13 antibody in infected rats, and reached high levels on days 45, 60 and 90 PI. In addition, the 28 kDa immunopositive bands were detected by anti-TIMP-1 antibody at all time points, and reached high levels on days 30, 45, 60 and 90 PI (Figure 1). Time-course studies of MMP-2 revealed that 72 kDa pro-MMP-2 bands were detected at all time points. Active forms of 64 kDa bands were detected on days 45, 60 and 90 PI. In addition, 21 kDa immunopositive bands were detected by anti-TIMP-2 antibody at all time points, and reached high levels on days 30, 45, 60 and 90 PI. The ratio of pro-MMP-2 to TIMP-2 decreased on days 60 and 90 PI. In contrast, the ratio of active-MMP-2 to TIMP-2 increased on days 45, 60 and 90 PI (Figure 2). The 440 kDa fibronectin dimer was present at all time points, showing significant degradation (present 220 kDa monomer) on days 45, 60 and 90 PI (Figure 3).

Figure 1.

Figure 1

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Protein levels of MMP-9, -13 and TIMP-1. (a) The 94 kDa pro-MMP-9 bands were significantly increased on days 45, 60 and 90 postinoculation (PI). The 60 kDa MMP-13 was present in infected groups, with significant increases on days 45, 60 and 90 PI. TIMP-1 was present at all time points, with significant increases on days 30, 45, 60 and 90 PI. β-actin was used as a loading control. (b) Quantitative analysis of MMP-9/TIMP-1 ratios were performed with a computer-assisted imaging densitometer system. The ratio levels of MMP-9 and TIMP-1 were significantly elevated (*P< 0.05) on days 45, 60 and 90 PI compared with uninfected control. (c) Quantitative analysis of MMP-13/TIMP-1 ratios were significantly elevated (*P< 0.05) in infected rats on days 45, 60 and 90 PI compared with uninfected control. MMP, matrix metalloproteinase; TIMP, tissue inhibitors of MMP.

Figure 2.

Figure 2

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Protein levels of MMP-2 and TIMP-2. (a) MMP-2 revealed that there were 72 kDa pro-MMP-2 bands detected at all time points. The active form of 64 kDa bands could be detected on days 45, 60 and 90 postinoculation (PI). In addition, 21 kDa immunopositive bands were detected by anti-TIMP-2 antibody at all time points, and reached high level on days 30, 45, 60 and 90 PI. (b) Quantitative analysis of MMP-2/TIMP-2 ratios were performed with a computer-assisted imaging densitometer system. The ratios of active MMP-2 and TIMP-2 were significantly elevated (*P< 0.05) in infected rats on days 45, 60 and 90 PI compared with uninfected control. MMP, matrix metalloproteinase; TIMP, tissue inhibitors of MMP.

Figure 3.

Figure 3

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Protein levels of fibronectin in the lung tissue. (a) The 440 kDa fibronectin dimer was present at all time points, showing significant degradation on days 45, 60 and 90 postinoculation (PI). The 220 kDa fibronectin monomer was increased on days 45, 60 and 90 PI. β-actin was used as a loading control. (b) Quantitative analysis of fibronectin were performed with a computer-assisted imaging densitometer system. The degradation of fibronectin was significantly elevated (*P< 0.05) in infected rats compared with uninfected control.

Interaction between fibronectin and MMPs

Western blot analysis showed that fibronectin was degraded from day 45 PI. Thus, we used this time point for the assay of interaction between fibronectin and MMPs. Immunoprecipitation of lung homogenates with anti-rat fibronectin antibody was followed by immunoblotting with anti-rat MMP-2, -9 or -13 antibodies. Results showed interaction between fibronectin and MMP-2, fibronectin and MMP-9, and fibronectin and MMP-13 (Figure 4).

Figure 4.

Figure 4

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Interaction between fibronectin and matrix metalloproteinases (MMPs). Immunoprecipitation of lung homogenates with anti-rat fibronectin antibody, was followed by immunoblotting with anti-rat MMP-2, -9 or -13 antibodies respectively. Results showed interaction between fibronectin and MMP-2 (a), fibronectin and MMP-9 (b), fibronectin and MMP-13 (c). The arrowheads indicate MMP bands. H, immunoglobulin heavy chain.

Localization of the MMP-13, TIMP-1 and -2 proteins

Histological changes were observed in the lungs of A. cantonensis-infected rats. Fibrotic granuloma showed dense circumferential accumulation of ECM components surrounding the granulomas and the first-stage larvae. On day 45 PI, MMP-13, TIMP-1 and -2 were detected in the lung tissue of A. cantonensis-infected rats. No positive signal could be detected with normal serum in the infiltrating leucocytes (Figure 5a). Positive signals for MMP-13 (Figure 5b), TIMP-1 (Figure 5c) and TIMP-2 (Figure 5d) could be detected in the infiltrating leucocytes.

Figure 5.

Figure 5

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Localization of MMP-13, TIMP-1 and -2 in the lung tissue. (a) No positive signal could be detected with normal serum in the infiltrating leucocytes of granuloma (G). Positive signals for MMP-13 (b), TIMP-1 (c) and TIMP-2 (d) stained brown in the infiltrating leucocytes (arrows). Blank arrows indicate first-stage larvae of Angiostrongylus cantonensis; arrowheads indicate leucocytes. MMP, matrix metalloproteinase; TIMP, tissue inhibitors of MMP.

Discussion

Human lung fibrosis and experimental animal models of pulmonary fibrosis are often associated with pulmonary inflammation characterized by accumulation of leucocytes such as macrophages, lymphocytes, and granulocytes (Crystal et al. 2002). Microbial infection is accompanied by infiltration of alveoli with inflammatory cells. Resolution of inflammation is associated with removal of microbial infection and return of tissue architecture to normal. In contrast, failure to resolve inflammation results in chronic recruitment, matrix deposition and fibroblast migration and proliferation, leading to loss of gas exchange capacity (Heasman et al. 2003). In this study, we examined and characterized the pathogenesis of rats infected with A. cantonensis that resulted in the development of lung inflammation and fibronectin degradation. The fibrotic response to A. cantonensis egg deposition and larvae invasion was marked in the lungs. Leucocytic infiltration is also a characteristic of granulomatous fibrosis and is believed to contribute, at least in part, to the fibrotic response.

Controlled ECM remodelling mediated by proteinases constitutes a physiological phenomenon whereas excessive proteinase expression results in pathological remodelling. Fibronectin is subject to degradation by a variety of proteinases, including plasminogen activator, plasmin, 72-kDa gelatinase, stromelysin, matrilysin, cathepsins and elastase (Werb 1989; Birkedal-Hansen et al. 1993). Our previous study also demonstrated that granulomatous fibrosis was strongly associated with MMP-2 and -9 in A. cantonensis-infected rat lungs (Hsu et al. 2005). Furthermore, the present studies demonstrated that fibronectin avidly binds MMPs (MMP-2, -9 and -13). This is characterized by increased activity of MMPs and degradation of the lung matrix.

Complement is the principal humoral effector system of inflammation, and its proteolytic cleavage fragments serve as ligands for specific receptors on inflammatory cells. These ligand-receptor interactions seem to be important in host resistance to both microbial and multicellular parasitic infections (Fearon & Wong 1983). Fibronectin is widely distributed in plasma, intercellular matrices and inflammatory exudates, and because it has multiple biological roles in cell-cell and cell-matrix interactions as well as phagocytosis, it is reasonable to expect its involvement in parasitic infections (Wyler 1987). Studies of the role of fibronectin in parasitic diseases have focused largely on the function of this molecule in invasion of host cells by intracellular parasitic protozoa Trypanosoma cruzi and Leishmania (Ouaissi et al. 1984; Wyler et al. 1985). Additionally, it has been demonstrated that fibronectin participates in the development of granulomatous inflammation and subsequent fibrosis induced by Schistosome eggs (Nishimura et al. 1985). Our results confirm and extend these findings: fibronectin was also involved in A. cantonesis-induced granulomatous fibrosis, and proteolytic enzymes participate in the degradation of fibronectin.

Matrix metalloproteinase activity is primarily regulated at three levels: transcriptional control, proteolytic cleavage of the pro-form to the active-form, and inhibition by physiological proteinase inhibitors, such as TIMP (Nagase & Woessner 1999). In the rat, MMP-1 is not present; its function is performed by MMP-13 (Quinn et al. 1990). The present study detected by Western blot the proteins MMP-2, -9, -13, and their inhibitors TIMP-1 and TIMP-2 in granulomatous reactions. Additionally, immunohistochemical observations showed localization of MMP-13, TIMP-1 and -2 within the infiltrating leucocytes. Our previous study (Hsu et al. 2005) also demonstrated that MMP-2 and -9 localized in the infiltrating leucocytes. However, the positive signals of proteins were weak or undetectable in the lung granulomatous fibrosis of rats on day 90 PI, and the possible reason was that the infiltrating leucocytes participated in the process of fibrosis. These MMPs efficiently degrade fibronectin, suggesting these proteinases are of crucial importance in processes requiring basement membrane disruption and, presumably, tissue infiltration by leucocytes, the pathologic substrate of chronic inflammatory diseases.

In conclusion, we hypothesized that, by cleaving ECM components, MMP-2, -9 and -13, which cleave fibronectin, could play a role in granulomatous fibrosis of the lungs caused by A. cantonensis. These results suggest that MMP-2, -9 and -13 may participate in fibronectin degradation of A. cantonensis-induced granulomatous fibrosis.

Acknowledgments

We wish to thank I-Ting Kuan and Chun-Hsien Wu, Department of Parasitology, Chung Shan Medical University, for assistance in this study.

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